RESUMO
The Sp7/Osterix transcription factor is essential for bone development. Mutations of the Sp7 gene in humans are associated with craniofacial anomalies and osteogenesis imperfecta. However, the role of Sp7 in embryonic tooth development remains unknown. Here we identified the functional requirement of Sp7 for dentin synthesis and tooth development. Sp7-null mice exhibit craniofacial dysmorphogenesis and are completely void of alveolar bone. Surprisingly, initial tooth morphogenesis progressed normally in Sp7-null mice. Thus the formation of alveolar bone is not a prerequisite for tooth morphogenesis. Sp7 is required for mineralization of palatal tissue but is not essential for palatal fusion. The reduced proliferative capacity of Sp7-deficient ectomesenchyme results in small and misshapen teeth with randomly arranged cuboidal preodontoblasts and preameloblasts. Sp7 promotes functional maturation and polarization of odontoblasts. Markers of mature odontoblast (Col1a, Oc, Dspp, Dmp1) and ameloblast (Enam, Amelx, Mmp20, Amtn, Klk4) are barely expressed in incisors and molar tissues of Sp7-null mice. Consequently, dentin and enamel matrix are absent in the Sp7-null littermates. Interestingly, the Sp7 expression is restricted to cells of the dental mesenchyme indicating the effect on oral epithelium-derived ameloblasts is cell-nonautonomous. Abundant expression of Fgf3 and Fgf8 ligand was noted in the developing tooth of wild-type mice. Both ligands were remarkably absent in the Sp7-null incisor and molar, suggesting cross-signaling between mesenchyme and epithelium is disrupted. Finally, promoter-reporter assays revealed that Sp7 directly controls the expression of Fgf-ligands. Together, our data demonstrate that Sp7 is obligatory for the differentiation of both ameloblasts and odontoblasts but not for the initial tooth morphogenesis. © 2018 American Society for Bone and Mineral Research.
Assuntos
Ameloblastos/citologia , Ameloblastos/metabolismo , Diferenciação Celular , Odontoblastos/citologia , Odontoblastos/metabolismo , Fator de Transcrição Sp7/metabolismo , Animais , Animais Recém-Nascidos , Calcificação Fisiológica , Proliferação de Células , Colágeno/metabolismo , Dentina/metabolismo , Desenvolvimento Embrionário , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Incisivo/crescimento & desenvolvimento , Incisivo/metabolismo , Incisivo/ultraestrutura , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese , Palato/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7/deficiência , Fator de Transcrição Sp7/genética , Células-Tronco/metabolismoRESUMO
The Runx2 transcription factor is critical for commitment to the osteoblast lineage. However, its role in committed osteoblasts and its functions during postnatal skeletogenesis remain unclear. We established a Runx2-floxed line with insertion of loxP sites around exon 8 of the Runx2 gene. The Runx2 protein lacking the region encoded by exon 8 is imported into the nucleus and binds target DNA but exhibits diminished transcriptional activity. We specifically deleted the Runx2 gene in committed osteoblasts using 2.3-kb col1a-Cre transgenic mice. Surprisingly, the homozygous Runx2 mutant mice were born alive. The Runx2 heterozygous and homozygous null were grossly indistinguishable from wild-type littermates at birth. Runx2 deficiency did not alter proliferative capacity of osteoblasts during embryonic development (E18). Chondrocyte differentiation and cartilage growth in mutants was similar to wild-type mice from birth to 3 months of age. Analysis of the embryonic skeleton revealed poor calcification in homozygous mutants, which was more evident in bones formed by intramembranous ossification. Runx2 mutants showed progressive retardation in postnatal growth and exhibited significantly low bone mass by 1 month of age. Decreased bone formation was associated with decreased gene expression of osteoblast markers and impaired collagen assembly in the extracellular matrix. Consequently, Runx2 mutant bones exhibited decreased stiffness and structural integrity. By 3 months of age, bone acquisition in mutant mice was roughly half that of wild-type littermates. In addition to impaired osteoblast function, mutant mice showed markedly decreased osteoclast number and postnatal bone resorption. Taken together, functional deficiency of Runx2 in osteoblasts does not result in failed embryonic skeletogenesis but disrupts postnatal bone formation.
Assuntos
Desenvolvimento Ósseo , Reabsorção Óssea/metabolismo , Diferenciação Celular , Proliferação de Células , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Osteoblastos/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Regulação da Expressão Gênica/genética , Homozigoto , Camundongos , Camundongos Knockout , Osteoblastos/patologiaRESUMO
Runx2 transcription factor is essential for the development of mineralized tissue, and is required for osteoblast commitment and chondrocyte maturation. Mice with global deletion of Runx2 exhibit complete failure of bone tissue formation, while chondrocyte-specific Runx2-deficient mice lack endochondral ossification. However, the function of Runx2 after commitment of mesenchymal cells to the osteoblast lineage remains unknown. Here, we elucidate the osteoblast-specific requirements of Runx2 during development of the tissue. Runx2 was deleted in committed osteoblasts using Cre-recombinase driven by the 2.3kbCol1a1 promoter. Surprisingly, Runx2(ΔE8/ΔE8) mice were born alive and were essentially indistinguishable from wild-type littermates. At birth, we failed to detect any alterations in skeletal patterning or extent of bone development in homozygous mutants. However, by 4 weeks of age, mutant mice showed obvious growth deficiencies, and weighed 20-25% less than sex-matched wild-type littermates. Micro-CT analysis of the hindlimb revealed a dramatic decrease of 50% in both cortical and trabecular bone volume compared with wild-type mice. Consistent with this observation, trabecular number and thickness were decreased by 51% and 21%, respectively, and trabecular space was increased by 2-fold in limbs of Runx2(ΔE8/ΔE8) mice. In addition to poor acquisition of bone mass, the average density of hydroxyapatite was markedly decreased in bone of Runx2(ΔE8/ΔE8) mice. Together, these findings demonstrate that loss of Runx2 activity in committed osteoblasts impairs osteoblast function, and that Runx2 is critical for postnatal, but not embryonic endochondral ossification.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Animais , Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos KnockoutRESUMO
Synthesis of cartilage by chondrocytes is an obligatory step for endochondral ossification. Global deletion of the Runx2 gene results in complete failure of the ossification process, but the underlying cellular and molecular mechanisms are not fully known. Here, we elucidated Runx2 regulatory control distinctive to chondrocyte and cartilage tissue by generating Runx2 exon 8 floxed mice. Deletion of Runx2 gene in chondrocytes caused failure of endochondral ossification and lethality at birth. The limbs of Runx2(ΔE8/ΔE8) mice were devoid of mature chondrocytes, vasculature, and marrow. We demonstrate that the C-terminus of Runx2 drives its biological activity. Importantly, nuclear import and DNA binding functions of Runx2 are insufficient for chondrogenesis. Molecular studies revealed that despite normal levels of Sox9 and PTHrP, chondrocyte differentiation and cartilage growth are disrupted in Runx2(ΔE8/ΔE8) mice. Loss of Runx2 in chondrocytes also impaired osteoprotegerin-receptor activator of NF-κB ligand (OPG-RANKL) signaling and chondroclast development. Dwarfism observed in Runx2 mutants was associated with the near absence of proliferative zone in the growth plates. Finally, we show Runx2 directly regulates a unique set of cell cycle genes, Gpr132, Sfn, c-Myb, and Cyclin A1, to control proliferative capacity of chondrocyte. Thus, Runx2 is obligatory for both proliferation and differentiation of chondrocytes.